Centrosomes are the primary microtubule organizing centers of animal cells and consist of a pair of centrioles surrounded by pericentriolar-material. Centrosomes are crucial for many fundamental processes, including cell polarity, nuclear positioning, spindle assembly, and cell migration. Thus, centrosome number must strictly be regulated both in somatic cells and in the germline. In proliferating cells, a single centrosome duplicates to give rise to two units that are inherited by daughter cells. During gametogenesis one of the parental centrosomes must be eliminated, so that only one centrosome is inherited by the zygote. In most metazoans centrioles are eliminated in the female germline, while they are maintained during spermatogenesis and are thus contributed strictly paternally to the zygote. The molecular basis underlying the differential inheritance of centrioles in the female and the male germline is not understood. My goal is to take advantage of the unique tools developed in C.elegans to study centrosome biogenesis and investigate the mechanisms regulating centriole inheritance in the germline. I propose to pursue three complementary approaches to dissect this process: 1.Analyze the mode, timing and candidate factors affecting centriole elimination during oogenesis 2. Characterize sas-1, a mutant affecting centriole maintenance during spermatogenesis 3.Conduct a functional genomic screen to identify novel genes inducing centriole elimination in the female germline.

Understanding the effect of spatial confinement on protein folding is directly relevant to biological processes such as folding in a chaperonin cavity or in a ribosome tunnel. In addition, macromolecular "crowding" effects in the cellular environment can significantly alter folding from what is observed in vitro. In certain limits, the protein behavior in such a crowded environment can be equivalently modeled as protein localization in an effective confining boundary. Although several previous studies have provided important insights into this problem, a quantitative understanding of the shift in folding / unfolding thermodynamics and kinetics as a function of the confinement characteristics such as dimensionality and size of the cavity and its interactions with the protein molecules and under different crowding conditions is lacking. Our goal in this project is twofold, (i) elucidate the effect of confinement on protein folding transition states and folding rates as a function of confinement size and interactions between the protein atoms and the confining walls and (ii) projection of the dynamics of our multi-dimensional systems onto suitable low-dimensional reaction coordinates (RCs) in order to understand better the underlying mechanism. To accomplish these goals, we plan to utilize a coarse-grained description of proteins which has already been found to be useful to describe several aspects of protein folding mechanisms. The successful outcome of this research will contribute towards our understanding of protein folding, association and aggregation under “in vivo” like conditions.

The synapse is a specialized cell-cell contact site between neurons or neurons and muscles, and precision of synapse formation is critical in the formation of a neural circuit. While previous studies have identified the molecules that are sufficient for synaptogenesis, little is known about molecular mechanisms that inhibit synaptogenesis with non-target cells. To understand how neurons inhibit synaptogenesis between axons and non-target cells, I will utilize the DA9 neuron in C. elegans, in which the presynaptic terminal is restricted to the specific domain in the axon by the local activity of Wnt and its receptor Fz. As this signaling does not appear to belong to any known Wnt signaling cascade, I will identify the downstream components through characterizing mutants that have a similar phenotype to wnt mutants in terms of localization of synaptic markers. Concurrently, I will try to elucidate how Wnt signaling inhibits synaptogenesis, based on two hypotheses: 1. Wnt inhibits synapse formation 2. Wnt stimulates synapse elimination. Stage specific expression or inactivation of Wnt will reveal which step Wnt actually regulates. I also plan on examining the relationship between Wnt signaling and SCF complex (E3 ubiquitin ligase) in synapse elimination, as SCF complex is required for synapse elimination in other neurons. As Wnt signaling is essential in normal human development, and its deregulation causes various diseases including neurological disorder, understanding the molecular mechanisms of inhibition of synaptogenesis by the novel Wnt pathway will contribute to therapeutic treatment of these diseases, as well as in understanding the development of the human brain.

Sec4p is a Rab GTPase required for transport of secretory vesicles from the Golgi to the plasma membrane in yeast. Sec4p localizes on the secretory vesicle and controls vesicle transport, vesicle tethering and vesicle fusion with plasma membrane. In each case, activation of Sec4p by its guanine nucleotide exchange factor Sec2p is necessary for the function. Ypt32p, another Rab that regulates the export of cargo from Golgi to the plasma membrane, is required to recruit Sec2p to the secretory vesicle. Thus, Sec2p links two different Rabs and constitute a Rab-GEF cascade. However, the regulation mechanism of these Rabs in the transport pathway is unclear. The host laboratory has shown that Sec2p also bind to Sec15p, a component of the exocyst complex implicated in vesicle tethering at the plasma membrane. Since Ypt32p and Sec15p bind to Sec2p in a competitive manner, the mechanism that triggers the change in Sec2p to switch from binding Ypt32p to binding Sec15p would be important in regulating the Rab-GEF cascade. Moreover, Sec2p localization relies on the production of phosphatidylinositol 4-phosphate (PI4P) in the Golgi by the phosphatidylinositol 4-kinase Pik1p. In this proposal, I will focus on the regulatory mechanism of Sec2p in the exocytic pathway. First, I plan to establish the roles of Ypt32p and Sec15p in Sec2p localization by identification of their binding sites in Sec2p. Second, I will examine the domain in Sec2p that binds PI4P and other phosphoinositides. Finally, I will test the effects of altering the subcellular phosphoinositide distribution on Sec2p localization and functions, and examine the role of Pik1p on Sec2p localization to the secretory vesicle.

Fanconi Anemia (FA) is a cancer susceptibility syndrome caused by mutations in genes encoding factors involved in replication-dependent removal of interstrand DNA crosslinks. Most FA members identified to date form a nuclear complex that ubiquitinates the proteins FANCD2 and FANCI in response to DNA damage, leading to formation of DNA repair foci. FA is thought to coordinate three different pathways: homologous recombination, nucleotide excision repair and mutagenic translesion synthesis (TLS) to remove crosslinks, but its mode of action is unclear. This research will study the FA-controlled repair process by focusing on identification and characterization of new members of the pathway. In order to understand how they promote mutagenic DNA synthesis, I will investigate if FA proteins recruit TLS enzymes directly, or otherwise I will identify the factors that connect them. For this, I will use both a proteomic approach to unravel protein complexes associated with TLS polymerases, and perform whole-genome RNAi screens to identify genes required for FA-dependent mutagenesis. Also, to study how the repair process is activated, I plan to uncover novel proteins recruited by ubiquitinated FANCD2-FANCI complexes, by purifying FANCD2 under conditions that stabilize the modified form. To assay if the candidates found by all these approaches are involved in the FA pathway, I will investigate if they exhibit the genetic and biochemical hallmarks of this pathway. This research is expected to reveal insights into the mechanism by which FA steers the repair of DNA crosslinks, and how deregulation of this essential genome maintenance mechanism leads to cellular transformation.

The transfer of mechanical energy via hollow structures is of great interest in many areas of engineering. Tubes as waveguides are matters of investigation since they are subject to many constraints, one being external acoustic radiation via the tube wall. In human hearing aids for example, acoustic radiation from tube walls causes feedback. Acoustic insects like bush-crickets detect sound with a system of paired ear drums on each foreleg, just below the knee. An air-filled tube, the acoustic trachea, delivers sound from the acoustic spiracle on the side of the body down the foreleg to the internal face of the eardrums. The wall structure of this tracheal tube is flexible and elastic, allowing sharp curvatures (produced by leg articulation) along its length. This ‘air tube’ is thus confined inside a fluid-filled chitin cylinder. The acoustic trachea of insects and human hearing aids tubing have remarkably analogous functions the former is however effective at much smaller length scales, opening up the opportunity of further miniaturisation of acoustic wave guides. This project aims at elucidating the mechanisms by which ears endowed with wave guides function to generate high sensitivity and directional accuracy. The role of accessory structures backing a conventional eardrum has been largely neglected thus far. Using mathematical modelling, experimental and theoretical data will be exploited to understand the modalities of sound propagation these auditory systems. The principles of sound propagation in biological wave guides may find direct applications for the development of novel detection technologies, notably in hearing aids and smart miniature pressure sensors.

Information coding in biological networks involves collective modes that cannot be simply described as the sum of their parts. For example, in the retina, these collective behaviors are governed by correlations that seem to be intrinsic to the statistical properties of the neuronal response, rather than determined by the stimulus. These correlations, albeit weak, produce strong collective effects with potential associative or error-correcting properties, reminiscent of spin-glasses and artificial communication systems. My proposal aims to understand this complex correlative structure and its implications for information encoding and processing. To this end I will use statistical models inspired from physics to analyze neural activity in the retina, as proposed by Scheidmann, Berry, Segev and Bialek. These models provide a unique opportunity to investigate theoretically, and test experimentally, the relevance of physical concepts to biological networks, among which the multiplicity of states, collective effects and robustness to error. Collaborating directly with experimentalists of Michael Berry's lab, I will work on new computational methods for inferring parameters from the data, and use these parameters to study the questions of false alarm rates and predictive feature detection. To address these problems I will apply some of the methods I have acquired as a graduate student working in the physics of disordered systems. I expect the fellowship to help me through my change of research area and, more generally, to help me learn about interesting problems and questions in quantitative biology.

The definitive hematopoietic stem cells (HSCs) competitively develop in the aorta-gonad-mesonephros and subsequently occupy stem cell niches in the definitive hematopoietic organs to ensure the long-term supply and homeostasis of mature blood cell types. Due to the lack of a genetically amenable readout system, little is known about the signaling pathways and processes involved in HSC development, migration, or niche colonization. This is also of clinical interest, as hematopoietic cancers develop upon oncogenic HSC transformation. Using zebrafish, we will develop a multi-fluorescence readout system to monitor HSC competition and development in descendant cells of HSCs, such as erythrocytes. For this purpose, we will construct two expression-vectors for erythrocyte-specific expression of GFP or RFP, respectively, and will additionally equip the GFP vector with a stem cell-specific expression cassette to co-overexpress modifier transgenes. Per default, erythrocytes with GFP and RFP fluorescence will appear in the zebrafish upon vector injection. However, if the modifier transgene positively affects HSC competitiveness, GFP-expressing cells will expand at the expense RFP-positive cells, thus revealing the competitive status upon modifier overexpression during HSC development and niche colonization. Our multi-fluorescence readout system will allow the focused study of candidate modifier genes and thus the analysis of vast array of questions concerning HSC development. Furthermore, system can be harnessed for screening of cDNA libraries to identify factors affecting HSC development and competition.

Embryogenesis is a robust and faithful process despite often significant perturbations in cellular movements and signaling processes. One of the most dramatic yet poorly understood examples of this robustness is the capability of early embryos to rebuild entire morphogenetic fields even when large parts of the embryo are removed. Despite its discovery more than 100 years ago, the mechanisms that underlie this phenomenon of self-regulation are almost completely unknown. To analyze how the embryo buffers developmental fluctuations, my postdoctoral research will explore the cellular and molecular mechanisms that govern self-regulation in zebrafish. First, I will characterize the cellular basis of self-regulation by analyzing the recovery of embryos after tissue removal at different developmental stages. Once I have defined the developmental window of self-regulation, I will test whether changes in cell fate or in cellular proliferation underlie this phenomenon. Finally, I will study the molecular basis of self-regulation by modulating the strength of developmental signaling pathways and by using a combination of genetics, chemical genomics and gene expression analysis.

The posttranslational modification (methylation, acetylation, phosphorylation, ubiquitination) of histones at specific residues regulates gene expression in eukaryotic genomes. Ultimately, such epigenetic marks, singly or in combination, decide cellular state and differentiated cell types. The significance of the histone code depends not only on the modification site but also the type and extent of modification. There exist several examples of reader proteins that recognize specific histone marks, which in turn recruit other proteins that facilitate changes in chromatin state. However no methyl-arginine (Rme) reader protein has been identified, even though it has been known that several arginine residues can be methylated. My first goal is to identify as many Rme reader proteins as possible by pull-down assays with synthesized Rme-containing peptides mimicking distinct histone-tail contexts. In addition, I will characterize the recognition mechanism and the extent of modification (mono- and dimethylation) of these modified-arginine peptides to identify the best substrates by structural and energetic characterization. My second goal is to understand the mechanism of combinatorial recognition of two or more histone marks (one of which is a Rme mark) in the context of the nucleosome as the template. The proposed studies should elucidate how arginine methylation, either singly or in combination with other marks, controls chromatin structure and function. My long-term goal is to broaden my structural expertise to functional experiments and use the proposed combined approach to contribute to the exciting developments in epigenetic regulation of the genome.

Adipose tissue plays an important role in energy homeostasis by producing proteins that influence glucose and lipid metabolism. Dysfunction of adipose tissue could lead to metabolic diseases, e.g., obesity, diabetes. Thus, studying the regulation of expression of these proteins would lead to better understanding how adipose tissue functions and possibly new therapeutic applications for metabolic disorders. Transcriptional regulation of genes involved in adipose function has been extensively studied. However, some observations suggested that the regulation could be more complex. Recent evidences also indicated that adipose function could be posttranscriptionally regulated. This proposal hypothesized that posttranscriptional control could modulate expression of genes involved in energy homeostasis and influence adipose function. Many genes involved in adipose function will be selected for this study, e.g., leptin, adiponectin. The experiments will investigate if posttranscriptional regulation is important for proper expression of these genes. And trans-factors (protein, RNAs) involved in this regulation will be isolated and identified, and their role will be verified. Finally, the significance of this regulation in adipose function will be examined in vivo. An emerging notion proposed that posttranscriptional regulation could co-ordinate expression of different genes, which have similar pattern of expression, by localizing the mRNAs in a translational inactive pool, and, upon certain stimuli, allowing the translation to take place. Apart from its appeal for practical use, this proposal also would, in practice, examine this possibility, which could have numerous implications.

Ca2+ waves are both necessary and sufficient for egg activation in animal fertilization, yet this is not known in plant fertilization though Ca2+ rises clearly occur in plant female gametes upon fusion with sperms. The objective of this proposed research is to test the hypothesis that Ca2+ wave can bypass the sperm-required fertilization in female gamete activation of flowering plants. Specifically, the study aims at profiling the cytosolic Ca2+ dynamics of plant female gametes that are activated dependently on or independently of fertilization. Live-imaged Ca2+ levels by two-photon (2P) microscopy will be reported by the yellow cameleon 3.6 protein expressed specifically in Arabidopsis female gametes of wild-type plants and several mutants having fertilization-independent gamete activation events. The results obtained from this study will furnish insights into the relationship between fertilization requirement and Ca2+ waves in plant gamete activation. They will also establish a foundation for subsequent investigations on the physiological responses of plant sexual and asexual reproduction modes. This fellowship will enable me to forge a new collaboration between my current home lab of Dr. Ueli Grossniklaus (Switzerland) and Dr. Seiji Takayama’s lab (Japan). As I will work together with the host lab to develop the new live-imaging 2P microscopy technique for physiological studies of plant fertilization processes, the fellowship will also assist my professional expansion of research approaches from the classical and molecular genetics to the new physiological level, and broaden my scientific perspectives in a different intellectual and cultural environment.

During adulthood, continuous production of blood cells is sustained by a population of hematopoietic stem cells (HSC) found inside bone marrow cavities. Self-renewing HSC differentiate into hematopoietic progenitor cells (HPC), which can give rise to all blood cell lineages (pluripotency). The specific microenvironments (niches) where HSC/P reside within BM architecture are likely to critically influence HSC/P fate and maintainance of the HSC compartment. Understanding the interactions that HSC/P need to establish with supporting elements found in specific niches is of crucial importance for transplantation biology. The chemokine CXCL12, as well as growth factors and integrins, have been shown to regulate HSC trafficking and engraftment. However, the intracellular signaling networks activated by these factors remain largely unknown. Focal adhesion kinase (FAK) plays a fundamental role in motility and survival of anchorage-dependent cells, such as fibroblasts. Previous studies indicate that FAK participates in CXCL12-induced signaling cascades required for HSC/P in vitro migration and adhesion. Here we propose to study the role of FAK in an in vivo setting by using gene-targeting inducible systems to specifically delete FAK expression in murine HSC. We will employ novel in vivo imaging techniques to analyze the impact of FAK deficiency in the ability of HSC to home to and engraft in BM niches. Furthermore, using a variety of in vivo and in vitro techniques we will dissect the roles of FAK in steady state hematopoiesis, HSC maintenance and lineage commitment.

Nemaline myopathy (NM) is an under-appreciated congenital neuromuscular disorder. NM is usually defined by severe muscle weakness. The overall frailty is handicapping for most of daily activities, and has a dramatic negative impact on the quality of life. To date, no cure exists. Therefore, the long-term scientific goals of our research are to (1) understand the mechanisms underlying weakness in NM; (2) develop sensitive, quick and accurate diagnostic tools; and (3) develop efficient intervention strategies. Recent advances demonstrate that NM is primarily due to mutations in genes coding muscle proteins and, in particular, proteins of the thin filament. Thin filament proteins mutations are typically missense mutations resulting in the substitution of just one amino acid in the proteins. This raises the question of the mechanisms by which such minor changes result in muscle weakness in NM. The present proposal specifically aims at investigating how thin filament proteins mutations alter thin filament structure and function leading to muscle weakness in NM. For this purpose, we plan to perform X-ray diffraction in skinned muscle fibres coming from biopsy sample specimens from patients carrying different mutations and from healthy controls. X-ray diffraction measures will be done in Japan because this specific method is not currently available in Sweden. The present project will constitute a significant component in our attempt to achieve our long-term goal and will allow for the translation of basic science into practical clinical knowledge. Therefore, we ask the Human Frontier Science Program for covering the present project.

Little is known about the role of visual experience during development and adulthood for high-level vision tasks like the fast recognition of faces and other objects. Studies have revealed many characteristics of object and face representations in the human and primate brain, but data on the role of visual experience in establishing and optimizing these representations are sparse and exploratory. I plan an innovative combination of studies to pinpoint how high-order representations of objects and faces are influenced by visual experience during development and adulthood. These studies include functional magnetic resonance imaging (fMRI) studies of object learning in healthy human adults, fMRI experiments in human patients with low visual acuity, and extracellular recordings in rats exploring how manipulations of visual input and its relevance during development and adulthood affect the characteristics and existence of high-order object representations. An important theme across these experimental lines is how the quality and the statistical structure of visual experience sets up a representation of objects and faces in terms of complex and regularly appearing conjunctions of more elementary features that are useful to distinguish between object categories and exemplars. In combination, these exciting studies have the potential to show what aspects of visual experience during development and adulthood help the brain to optimize its representations of the faces and other objects in the world. Support through the HFSP CDA will be invaluable to help me implement this line of work.

To ensure their proper function throughout adult life, epithelial tissues balance cell growth, differentiation, and apoptosis. Homeostasis aberration leads to malfunction of tissues and severe diseases, including cancer. TGFb signaling has a significant role in the regulation of homeostasis. As an intriguing property, TGFb signaling is negative regulator of epithelial cell proliferation, whereas it can promote invasion and metastasis during the later stages of tumor progression. Surprisingly, epithelia lacking the essential receptor (TbRII) for TGFb signaling display morphologically normal tissue homeostasis by balancing hyperproliferation and elevated integrin-signaling with elevated apoptosis. However, this balance can be easily disrupted with additional homeostasis challenges. These mice display accelerated wound-repair and increased propensity to form squamous cell carcinomas (SQCCs). To elucidate the detailed molecular mechanisms underlying epithelial homeostasis downstream of TbRII, I will identify up/downregulated genes and posttranslational modifications arising from epidermal TGFb signaling and examine how these changes are affected in TbRII null vs WT epithelia, when subjected to wound-repair and SQCC progression. I will employ an ShRNA screening strategy to evaluate how these changes affect proliferation, apoptosis, cell migration and invasion in keratinocytes in vitro, and employ skin engraftment of ShRNA-infected keratinocytes in vivo and mouse genetics to determine the physiological relevance of my findings. These studies will advance our understanding of tissue homeostasis and may provide useful clinical methods for treatment of wound-healing and cancer.

Recent observations demonstrated that leptin, an adipocyte-derived hormone, is a major regulator of bone remodeling through the sympathetic nervous system. However, the molecular mechanisms used by leptin to regulate sympathetic activity that inhibits bone formation remain elusive. My sponsor’s laboratory is studying the influence of neuropeptides on bone formation, whose expression is regulated by leptin. Among these, serotonin is an interesting candidate, because in patients serotonin reuptake inhibitors affect bone remodeling. 5-Ht2c is a serotonin receptor most highly expressed in the hypothalamus and 5-Ht2c -/- mice display a low bone mass due to a decrease in bone formation. Furthermore, expression of Tph2 (an enzyme responsible for serotonin production in the brain) and serotonin levels in the hypothalamus, are both elevated in leptin-deficient mice. These observations suggest that serotonin acts downstream of leptin and upstream of sympathetic activity to regulate bone formation. To test this hypothesis, I will generate Tph2-Egfp Knock-in mice to assess whether Tph2 regulates bone mass and sympathetic activity. Using axon tracing, I will test if axonal projections of Tph2-expressing neurons target 5-Ht2c-expressing neurons in the hypothalamus. Finally, I propose to genetically and physiologically determine whether leptin and serotonin act in the same pathway regulating bone formation.

Cell fate and pattern formation are fundamental developmental processes in all multicellular organisms. The formation of stomata in plants constitutes an excellent model to study basic developmental question and molecular mechanisms underlying these processes. Several regulators of stomatal development have been described. Among the most prominent is the SPEECHLESS (SPCH) gene, since it is necessary for initiation of the entire stomatal formation program. Ensuring that SPCH is active at the correct time and place is essential, yet there is no data available about how SPCH activity is modulated. My first aim is to identify regulators of the SPCH gene and protein. Based on our observations that SPCH activity is de-repressed in certain mutant backgrounds, I will screen for and characterize additional negative regulators of SPCH using both a candidate gene and unbiased mutagenesis approach. My second aim is to investigate the precise role of a newly identified regulator of stomata development. The mutant phenotype is characterized by abnormal epidermal cell divisions which result in formation of aberrant clusters of cells and stomata in contact. Through physiological and molecular analysis I will investigate the function of this gene, where and when it is required, and its connection to known stomata regulators.